title: A highly sensitive in vivo footprinting technique for condition-dependent identification of cis elements.
abstract: Knowing which regions of a gene are targeted by transcription factors during induction or repression is essential for understanding the mechanisms responsible for regulation. Therefore, we re-designed the traditional in vivo footprinting method to obtain a highly sensitive technique, which allows identification of the cis elements involved in condition-dependent gene regulation. Data obtained through DMS methylation, HCl DNA cleavage and optimized ligation-mediated PCR using fluorescent labelling followed by capillary gel electrophoresis are analysed by ivFAST. In this work we have developed this command line-based program, which is designed to ensure automated and fast data processing and visualization. The new method facilitates a quantitative, high-throughput approach because it enables the comparison of any number of in vivo footprinting results from different conditions (e.g. inducing, repressing, de-repressing) to one another by employing an internal standard. For validation of the method the well-studied upstream regulatory region of the Trichoderma reesei xyn1 (endoxylanase 1) gene was used. Applying the new method we could identify the motives involved in condition-dependent regulation of the cbh2 (cellobiohydrolase 2) and xyn2 (endoxylanase 2) genes.
authors.affiliation: Research Division Biotechnology and Microbiology, Institute of Chemical Engineering, Vienna University of Technology, Gumpendorfer Str. 1 a, A-1060 Vienna, Austria and Department of Biology, University of Ottawa, Gendron Hall, 30 Marie Curie, Ottawa, ON, K1N6N5, Canada.
authors.forename: Rita
authors.initials: R
authors.lastname: Gorsche
authors.forename: Birgit
authors.initials: B
authors.lastname: Jovanovic
authors.forename: Loreta
authors.initials: L
authors.lastname: Gudynaite-Savitch
authors.forename: Robert L
authors.initials: RL
authors.lastname: Mach
authors.forename: Astrid R
authors.initials: AR
authors.lastname: Mach-Aigner
mesh_terms: D043366:Cellulose 1,4-beta-Cellobiosidase / Q000235:genetics; D053837:DNA Cleavage; D018983:DNA Footprinting / Q000379:methods*; D019175:DNA Methylation; D043364:Endo-1,4-beta Xylanases / Q000235:genetics; D016133:Polymerase Chain Reaction; D050436:Regulatory Elements, Transcriptional; D012984:Software; D014242:Trichoderma / Q000235:genetics
chemical_list: D043364:Endo-1,4-beta Xylanases; D043366:Cellulose 1,4-beta-Cellobiosidase